Characterization of Gastrointestinal Drug Absorption in Cynomolgus

Feb 2, 2008 - found to show the low fraction absorbed into portal vein (FaFg), suggesting that the low BA in cynomolgus monkeys was attributed mainly ...
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articles Characterization of Gastrointestinal Drug Absorption in Cynomolgus Monkeys Masayuki Takahashi,*,† Takuo Washio,† Norio Suzuki,† Katsuhiro Igeta,† Yoshimine Fujii,† Masahiro Hayashi,‡ Yoshiyuki Shirasaka,§ and Shinji Yamashita§ Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan, Faculty of Pharmaceutical Science, Tokyo UniVersity of Pharmacy and Life Science, Tokyo, Japan, and Faculty of Pharmaceutical Science, Setsunan UniVersity, Osaka, Japan Received July 19, 2007; Revised Manuscript Received November 28, 2007; Accepted November 28, 2007

Abstract: Possible factors of species differences in gastrointestinal drug absorption between cynomolgus monkeys and humans were examined using several commercial drugs. Oral bioavailability (BA) of acetaminophen, furosemide, and propranolol in cynomolgus monkeys was significantly lower than that in humans. From the pharmacokinetic analysis, these drugs were found to show the low fraction absorbed into portal vein (FaFg), suggesting that the low BA in cynomolgus monkeys was attributed mainly to the gastrointestinal absorption processes. The gastric emptying rate (GER) calculated from plasma concentration profiles after oral administration of acetaminophen in cynomolgus monkeys was similar in humans. The gastrointestinal transit time (GITT) in cynomolgus monkeys was only slightly shorter than that in humans. On the other hand, it was demonstrated that the apparent intestinal permeability (Papp) of five drugs to cynomolgus monkey intestine was lower than that to rat intestine; especially propranolol and furosemide showed the remarkably low Papp. The expression levels of mRNAs of efflux transporters analyzed by real-time RT-PCR indicated that mRNA expression levels of MDR1, MRP2, and BCRP in monkey intestine were significantly higher than those in human intestine. This result suggested that low oral absorption of furosemide in cynomolgus monkeys was attributed to the high activities of efflux transporters in its intestinal membrane. Results of in vivo PK analysis clearly showed that FaFg values of propranolol and acetaminophen in cynomolgus monkeys were markedly lower than those in humans. Since propranolol and acetaminophen were the drug with high membrane permeability, it was considered that the high first-pass metabolism in the enterocytes was a main factor of their low FaFg in cynomolgus monkeys. In conclusion, it was demonstrated that the high activities of efflux transporters and/ or metabolizing enzymes in the intestinal membrane are possible factors to cause poor oral absorption of drugs in cynomolgus monkeys. Keywords: Cynomolgus monkey; species difference; oral bioavailability; intestinal permeability; gastrointestinal physiology; Ussing-type chamber

Introduction Rats, dogs and monkeys are often used for pharmacokinetics (PK) studies during the drug discovery and * Corresponding author. Mailing address: Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan. Tel: 81-3-3492-3131. Fax: 81-3-5436-8567. E-mail: [email protected]. 340

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development process in the pharmaceutical industries. Since oral bioavailability (BA) is a key factor for development of oral new chemical entities, the oral BA in human should be predicted accurately to enhance the success probability in the clinical study. However, these †

Daiichi Sankyo Co., Ltd. Tokyo University of Pharmacy and Life Science. § Setsunan University. ‡

10.1021/mp700095p CCC: $40.75  2008 American Chemical Society

Published on Web 02/02/2008

Gastrointestinal Drug Absorption in Cynomolgus Monkeys experimental animals sometimes show significant species differences in the oral BA of drugs that might lead to the erroneous prediction of the BA in humans. Especially in monkeys, remarkably low oral BA has often been shown even if the high BA was shown in other species for the same drugs. In general, oral BA of drugs can be expressed as BA ) FaFgFh where Fa is a fraction dose absorbed from the gastrointestinal tract, Fg is intestinal availability (a fraction not metabolized in the gut wall), and Fh is a hepatic availability (a fraction not metabolized in the liver). Species differences in oral BA are attributed to any of these processes in oral drug absorption. In order to improve the predictability of drug BA in humans from the preclinical animal studies, it is important to determine the factors involved in the species differences in oral drug absorption. Chiou et al. have carefully surveyed the literature and calculated Fa of various drug in rats,1 dogs,2 and cynomolgus monkeys3 and compared them with that in humans. Since Fa in animals correlated well with that in humans, they have concluded that species differences were not significant in Fa. In contrast, insufficient correlations in drug absorption between experimental animals and humans were reported due to the differences in gastric pH4–6 and/or the gastric emptying rate and gastrointestinal motility.7–11 Also, the differences in the activity of influx such as PepT1 or efflux transporters such as MDR1 might cause the species differences in Fa. Differences in the activity of metabolic enzymes in GI tract and liver cause the species differences in Fg or Fh. Chiou et al. have also shown the higher nonrenal clearance in monkeys than that in humans and thus concluded that low oral BA in cynomolgus monkeys is mainly attributed to the low Fh of drugs due to the high first-pass metabolism in the liver.3 Contrary to this report, Ward and Smith have identified the clearance of 103 drugs in rats, dogs, monkeys, and humans after intravenous administration and evaluated the (1) Chiou, W. L.; Barve, A. Linear correlation of the fraction of oral dose absorbed of 64 drugs between humans and rats. Pharm. Res. 1998, 15, 1792–1795. (2) Chiou, W. L.; Jeomg, H. Y.; Chung, S. M.; Wu, T. C. Evaluation of using dogs as an animal model to study the fraction of oral dose absorbed of 43 drugs in humans. Pharm. Res. 2000, 17, 135– 140. (3) Chiou, W. L.; Buehler, P. W. Comparison of oral absorption and bioavailability of drugs between monkey and human. Pharm. Res. 2002, 19, 868–874. (4) Lui, C. Y.; Amidon, G. L.; Berardi, R. R.; Fleisher, D.; Youngberg, C.; Dressman, J. B. Comparison of gastrointestinal pH in dogs and humans: implications on the use of the beagle dog as a model for oral absorption in humans. J. Pharm. Sci. 1986, 75, 271–274. (5) Yamada, I.; Goda, T.; Kawata, M.; Shiotoki, T.; Ogawa, K. Gastric acidity-dependent bioavailability of commercial sustained release preparations of indomethacin, evaluated by gastric aciditycontrolled beagle dogs. Chem. Pharm. Bull. 1990, 38, 3112–3115. (6) Kanarva, H.; Klebovich, I.; Drabant, S.; Urtti, A.; Nevalainen, T. Different absorption profiles of deramciclane in man and in dog. J. Pharm. Pharmacol. 1998, 50, 1087–1093.

articles predictability of each animal for human drug clearance.12 In all instances, the monkey tended to provide the most qualitatively and quantitatively accurate predictions of human clearance and also afforded the least biased prediction compared with other species. According to this report, Fh in monkeys is not necessarily higher than that in humans. In contrast, several reports have demonstrated the high metabolic activity of monkey intestine for CYP3A4 substrate drugs. In the case of midazolam, a typical substrate of CYP3A, FaFg value in cynomolgus monkeys was calculated as 0.03–0.113,14 and markedly lower than that in humans (approximately 0.5). Although the exact value of Fg was not calculated, the low oral absorption of midazolam in cynomolgus monkeys was considered to be attributed to the high first-pass metabolism in the intestine.13 Also, it was reported that the oxidation activities and conjugation activities in the cynomolgus monkey intestine were higher than those in human intestine.15,16 These findings have implied the possibility of high intestinal first-pass metabolism in monkeys for various drugs. Therefore, as the test drugs in this study, we have included the substrates of CYP (CYP2D6) and UGT-glucuronosyltransferase (UGT), such as propranolol and acetaminophen, respectively, in addition to the drugs with high and low permeability to the intestinal membrane (7) Mizuta, H.; Kawazoe, Y.; Ogawa, K. Gastrointestinal absorption of chlorothiazide: Evaluation of a method using salicylazosulfapyridine and acetaminophen as the marker compounds of the gastrointestinal transit time in beagle dog. Chem. Pharm. Bull. 1990, 38, 2810–2813. (8) Dressman, J. B. Comparison of canine and human gastrointestinal physiology. Pharm. Res. 1986, 3, 123–131. (9) Katori, N.; Aoyagi, N.; Kojoma, S. Effects of codeine on the agitating force and gastrointestinal transit time in dogs, for use in drug absorption studies. Biol. Pharm. Bull. 1998, 21, 418–420. (10) Kondo, H.; Takahashi, Y.; Watanabe, T.; Yokohama, S.; Watanabe, J. Gastrointestinal transit of liquids in unfed cynomolgus monkeys. Biopharm. Drug Dispos. 2003, 24, 131–140. (11) Ikegami, K.; Tagawa, K.; Narisawa, S.; Osawa, T. Suitability of the cynomolgus monkey as an animal model for drug absorption studies of oral dosage forms from viewpoint of gastrointestinal physiology. Biol. Pharm. Bull. 2003, 26, 1442–1447. (12) Ward, K. W.; Smith, B. R. A comprehensive quantitative and qualitative evaluation of extrapolation of intravenous pharmacokinetic parameters from rat, dog, and monkey to humans. I. Clearance. Drug Metab. Dispos. 2004, 32, 603–611. (13) Sakuda, S.; Akabane, T.; Teramura, T. Marked species difference in the bioavailability of midazolam in cynomolgus monkeys and humans. Xenobiotica 2006, 36, 331–340. (14) Nishimura, T.; Amano, N.; Kubo, Y.; Ono, M.; Kato, Y.; Fujita, H.; Kimura, Y.; Tsuji, A. Asymmetric intestinal first-pass metabolism causes minimal oral bioavailability of midazolam in cynomolgus monkey. Drug Metab. Dispos. 2007, 35, 1275–1284. (15) Prueksaritanont, T.; Gorham, M. L.; Hochman, H. J.; Tran, O. L.; Vyas, K. Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in caco-2 cells. Drug Metab. Dispos. 1996, 24, 634–642. (16) Kaji, H.; Kume, T. Glucuronidation of 2-(4-chlorophenyl)-5-(2furyl)-4-oxazoleacetic acid (TA-1801A) in humans: species differences in liver and intestinal microsomes. Drug Metab. Pharmacokinet. 2005, 20, 206–211. VOL. 5, NO. 2 MOLECULAR PHARMACEUTICS 341

articles (naproxen and atenolol).17,18 Furosemide was also included as a substrate of efflux transporters. In the present study, in order to consider the first-pass metabolism in cynomolgus monkeys, PK parameters of several drugs were calculated from the time-course of plasma concentration after intravenous and oral administration. Since Fa and Fg are obtained as a hybrid (FaFg) and cannot be isolated only from the in ViVo data, we have measured the intestinal permeability of drugs in cynomolgus monkeys by the in Vitro Ussing-type chamber method. Lennernäs et al. have demonstrated a good correlation between the effective permeability of the human jejunum obtained with in ViVo single pass perfusion and that of rat jejunum obtained with the Ussingtype chamber method.19 Therefore, it seems that the species differences in Fa can be evaluated by measuring the membrane permeability in Vitro. In addition, GI transit time and expression levels of mRNAs of the efflux transporters in cynomolgus monkeys were evaluated and compared with those in humans to elucidate its effects on Fa.

Materials and Methods Materials. Acetaminophen, salitylazosulfapyridine, sulfapyridine, propranolol, atenolol, furosemide, naproxen, p-anisamide, and warfarin were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). D82-7319, an internal standard for the LC/MS/MS analysis, was synthesized by the Medicinal Chemistry Research Laboratory, Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). All other chemicals were obtained from commercial sources and were of analytical or the highest available grade. In ViWo Pharmacokinetic Studies in Monkeys. Female cynomolgus monkeys weighing 2.5–3.5 kg were used. Cynomolgus monkeys (n ) 3–5) were fasted overnight prior to drug administration, whereas access to water was provided ad libitum. Each compound was suspended in 0.5% methylcellulose solution and administered orally with a gastric tube. The intravenous dose was injected in the forearm vein as saline solution. The cynomolgus monkeys were restricted to a chair only at the time of drug administration and blood collection. Approximately 1 mL blood samples were collected using heparinized syringes at the designated time intervals after drug administration via the femoral vein. Plasma was separated by centrifugation of the blood sample at 3000 rpm (1800g) for 10 min at 4 °C and stored at -20 °C until analysis. (17) Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995, 12, 413–420. (18) Fagerholm, U.; Johansson, M.; Lennernäs, H. Comparison between permeability coefficients in rat and human jejunum. Pharm. Res. 1996, 13, 1336–1342. (19) Lennernäs, H.; Nylander, S.; Ungell, A. L. Jejunal permeability: A comparison between the ussing chamber technique and the single-pass perfusion in humans. Pharm. Res. 1997, 14, 667–671. 342

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Takahashi et al. Evaluation of the Gastrointestinal Transit Time (GITT) in Cynomolgus Monkeys. Female cynomolgus monkeys weighing 2.5–3.5 kg (n ) 5) were fasted overnight, and 100 mg of salitylazosulfapyridine was administered with a gastric tube. Approximately 1 mL blood samples were collected using heparinized syringes every hour after administration for 8 h. The time of appearance of sulfapyridine in plasma after oral administration of salitylazosulfapyridine was evaluated. Plasma sulfapyridine concentrations were analyzed by HPLC according to the method of Mizuta et al.7 The average time of appearance of sulfapyridine in plasma of five cynomolgus monkeys was used as GITT. Evaluation of Blood to Plasma Concentration Ratio (Rb). Each drug solution (100 µg/mL in 20% methanol solution) was diluted with 20% methanol solution, and finally a 2 µg/mL solution of each compound was prepared. Forty microliters of each drug solution was added to 360 µL of blank blood, and it was incubated for 10 min at 37 °C. The mixture was centrifuged at 3000 rpm (1800g) for 10 min at 4 °C. To 100 µL aliquots of supernatant, 50 µL of IS (warfarin, 200 ng/mL) acetonitrile solution and 50 µL of acetonitrile were added. The supernatant was transferred to MultiScreen (GV, 0.22 µm, Millipore, Billerica, MA) and filtered by centrifugation at 3000 rpm (1800g) for 10 min at 4 °C. Each filtrate was transferred to autosampler vials, and 10 µL portions were injected into the LC/MS/MS system. The Rb value was calculated by the following equation Rb ) Cb/Cp (1) where Cb is the plasma concentration when the compound is added to plasma and Cp is the plasma concentration when the compound is added to blood. In Vitro Intestinal Permeability. Membrane permeability was evaluated by the Ussing-type chamber method according to the previous reports.19,20 Experiments were performed in modified Ringer solution (pH 7.4) of the following composition: NaCl, 125 mM; KCl, 5 mM; CaCl2, 1.4 mM; NaH2PO4, 1.2 mM; NaHCO3, 10.0 mM; and D-glucose 11.1 mM in an atmosphere of a 95% O2/5% CO2 gas. The small intestinal tract of cynomolgus monkey was removed under light anesthesia with ketamine. A portion of the jejunum segment from a cynomolgus monkey was removed the muscle layer and was mounted to the Ussing-type chamber. The viability of tissue was checked by the permeability of sulfasalazine, the paracellular maker, and membrane conductance (data not shown). The initial drug concentration on the mucosal side was set at 0.1 mM for all compounds. Samples (100 µL) were withdrawn from both sides every 20 min up to 120 min. The apparent permeability coefficient (Papp) across the excised jejunum segment was calculated using the following equation: Papp ) dXR/dT × 1/A × C0 (2) where dXR/dT is the steady-state appearance rate on the (20) Watanabe, E.; Takahashi, M.; Hayashi, M. A possibility to predict the absorbability of poorly water-soluble drugs in human based on rat intestinal permeability assessed by an in vitro chamber method. Eur. J. Pharm. Biopharm. 2004, 58, 659–665.

Gastrointestinal Drug Absorption in Cynomolgus Monkeys serosal side, A is the exposed membrane surface area, and C0 is the initial concentration on the mucosal side. All animal experiments were conducted with the approval of the Animal Experiment Ethics Committee of Daiichi Pharmaceutical Co., Ltd. Pharmacokinetic Analysis. The area under the concentration-time curve (AUC), total body plasma clearance (CLtot.plasma), and the mean residence time (MRT) after intravenous and oral administration were calculated using the noncompartmental method without extrapolation using a validated program developed in-house. Oral BA was calculated as the ratio of AUC after oral and intravenous administration. Hepatic clearance (CLh), Fh, and FaFg were calculated using eqs 3-6 according to the theory of Rowland.21 The Rb of each drug was experimentally measured as described above. The hepatic blood flow (Qh) in cynomolgus monkeys (43.6 mL/min/kg) used the reported value.22 In the calculation, the urinary excretion ratios (fe) of each drug in cynomolgus monkeys was assumed to be the same as those in humans,23 0% for acetaminophen, naproxen and propranolol, 65% for furosemide, and 94% for atenolol, respectively. CLtot.blood ) CLtot.plasmaCL/Rb

(3)

CLh ) CLtot.blood(1 - fe)

(4)

Fh ) 1 - CLh/Qh

(5)

FaFg ) BA/Fh

(6)

The gastric emptying rate (GER) of cynomolgus monkeys was estimated by means of mean absorption time (MAT) calculated as follows MAT ) MRTpo - MRTiv

(7)

where MRT is the mean residence time after oral or intravenous administration of acetaminophen in cynomolgus monkey. The function that indicates a relationship between the Papp of drugs and FaFg in humans and cynomolgus monkeys is described by eq 8. FaFg ) 1 - exp(-Papp f)

(8)

The relationship between Papp and FaFg, which is expressed as the correction factor f, was determined by nonlinear leastsquares regression analysis (Levenbeg-Marquardt leastsquares algorithm; Delta Graph 4.0 by SPSS, Inc., Chicago, IL). (21) Rowland, M.; Tozer, T. N. Clinical Pharmacokinetics; Concept and Application; Lea & Febiger: Philadelphia, 1980; 2 (22) Davies, B.; Morris, T. Physiological parameters in laboratory animals and humans. Pharm. Res. 1993, 10 (7), 1093–1095. (23) Goodman, L. S.; Hardman, J. G.; Limbird, L. E. Goodman & Gilman’s the Pharmacological Basis of Therapeutics; 10th ed., Japanese ed.; Hirokawa Publishing Co.: Tokyo, 2003. (24) Nishimura, M.; Naito, S. Tissue-specific mRNA expression profiles of human ATP-binding cassette and solute carrier transporter superfamilies. Drug Metab. Pharmacokinet. 2005, 20, 452–477.

articles HPLC Analysis. Acetaminophen and sulfapyridine concentrations in plasma were measured by HPLC according to the methods of Mizuta et al.7 A liquid chromatograph (Model 600E, Waters, Milford, MA) equipped with UV detector (Model 484, Waters, Milford, MA), autosampler (Model 717, Waters), and autointegrator (Millennium, Waters) was used. For the stationary phase, a reversed-phased column (L-column ODS, 4.6 mm i.d. × 150 mm length, Chemicals Inspection and Testing Institute, Tokyo, Japan) warmed to 40 °C was used. The mobile phase was a mixture of 50 mM phosphate buffer adjusted to pH 6.8 and acetonitrile (100:14) containing 5 mM octylamine. The flow rate was 1.0 mL/min. Sample Preparation for HPLC Analysis. For the analysis of plasma concentrations of acetaminophen and sulfapyridine, 1 mL of 50 mM phosphate buffer, 0.1 mL of IS solution (p-anisamide, 20 µg/mL), and 2 mL of ethyl acetate were added to 0.2 mL of plasma. The mixture was shaken for 10 min and centrifuged at 3000 rpm (1800g) for 10 min at 4 °C. The organic layer was evaporated to dryness under reduced pressure. The residue was dissolved with mobile phase and transferred to autosampler vials, and a 10 µL portion was injected onto the HPLC system. Standard working solutions (10 µL) were added to blank plasma to give standard calibration samples at concentrations of 200–5000 ng/mL. LC/MS/MS Analysis. Concentrations of propranolol, atenolol, furosemide, naproxen in plasma and Ussing-type chamber medium, and acetaminophen in Ussing-type chamber medium were measured by the LC/MS/MS method consisting of Quattro Ultima (Waters) with Alliance 2695 separation module (Waters). Multiple reaction monitoring (MRM) mode was used as follows to monitor ion (precursor ion f product ion): atenolol (267.0 f 144.8), acetaminophen (152.2 f 109.8), propranolol (260.0 f 182.8), furosemide (329.07 f 285.04), naproxen (230.88 f 184.93), and D82–7319 (IS, 488.2 f 350.2). Samples were injected into a Symmetry Shield RP8 column (3.5 µm φ, 2.1 mm × 30 mm i.d., Waters) with Symmetry Shield RP8 guard column (3.5 µm φ, 2.1 mm × 10 mm i.d., Waters) warmed to 40 °C. Elution was conducted at a flow rate of 0.5 mL/min by a linear gradient with the mobile phase, which consisted of a mixture of A (10 mM ammonium formate solution) and B (methanol). The gradient conditions for elution were as follows: gradient [min, B%] ) [0, 30]-[0.5, 30]-[1.5, 90]-[3.5, 90]-[4, 30]-[5, 30]. Sample Preparation for LS/MS/MS Analysis. Methanol (150 µL) containing IS (D82-7319, 62.5 ng/mL) was added to the plasma and medium samples (50 µL) of Ussing-type chamber experiments and centrifuged at 3000 rpm (1800g) for 10 min at 4 °C. The supernatant was transferred to MultiScreen and filtered by centrifugation at 3000 rpm (1800g) for 10 min at 4 °C. The filtrate was transferred to autosampler vials, and a 10 µL portion was injected onto the LC/MS/MS system. Standard working solutions (10 µL) were added to blank plasma or medium to give standard VOL. 5, NO. 2 MOLECULAR PHARMACEUTICS 343

articles

Takahashi et al.

Table 1. Gene-Specific Primers of Monkey for SYBR Green PCR gene

forward primer

reverse primer

product size (bp)

GAPDH MDR1 BCRP MRP2

5′-ATTCCACCCATGGCAAGTTC-3′ 5′-AGCGGCTCCGATACATGGT-3′ 5′-AGCGGGATAAGCCACTCGTA-3′ 5′-ATGGCAGTGAAGAAGAAGACGAT-3′

5′-ACGTACTCAGCGCCAGCAT-3′ 5′-GGCGAGCCTGGTAGTCAAT-3′ 5′-CTCACCCCCGGAAAGTTGAT-3′ 5′-TGCTGCTGGACCTAGAACTG-3′

136 101 101 138

calibration samples at concentrations of 1–5000 ng/mL for LC/MS/MS. Data Analysis of HPLC and LC/MS/MS Data. The peak area of the compound was divided by the peak area of the IS to obtain the peak-area ratio. The calibration curve for the compound was constructed from the least-squares linear regression of the peak-area ratios of standards versus the compound concentrations. RNA Extraction and RT Reaction. Segments of jejunum, colon, and liver tissues were isolated from cynomolgus monkeys (n ) 7) and were quickly stripped of connective tissue, snap-frozen, and stored at -80 °C until processing. Total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen Japan K.K., Tokyo, Japan) and chloroform. The RT reaction was conducted in 10 µL of two-step RT reaction mix containing 2 µL of the extracted total RNA (200 µg/mL), 1 × TaqMan RT buffer, 5.5 mM MgCl2, 500 µM dATP, 500 µM dGTP, 500 µM dCTP, 500 µM dUTP, 2.5 µM oligo (dT)16 primer, 0.4 U/µL of Rnase inhibitor, and 1.25 U/µL MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). The mixture was incubated at 25 °C for 10 min and subsequently at 48 °C for 30 min. The RT reaction was terminated by heating at 95 °C for 5 min followed by cooling at 4 °C for 5 min, giving the RT product. Real-Time PCR with SYBR Green. Primer pairs for MDR1, BCRP, MRP2, and GAPDH were designed using the Primer Express (Ver. 1.0, Applied Biosystems) as shown in Table 1 and were synthesized by Invitrogen (Tokyo, Japan). A commercial reagent, QuantiTect SYBR Green PCR kit (QIAGEN, Hilden, Germany), was used for PCR. Each reaction mixture contained 300 nM each primer, 6.5 µL of RT product, and SYBR Green PCR Master Mix (QIAGEN) in a total volume of 25 µL. PCR conditions were 10 min at 95 °C followed 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The relative increase of the reporter fluorescent dye emission was monitored in real time using an ABI prism 7000 sequence detector (Applied Biosystems). The fluorescent dye emission was a function of cycle number and was determined using the sequence detector software (Applied Biosystems), giving the threshold cycle number (CT) at which PCR amplification reached a significant threshold. The mRNA levels of MDR1, BCRP, and MRP2 are expressed as values relative to GAPDH mRNA.

Results The oral BA of five drugs in cynomolgus monkeys are summarized in Table 2 with that in humans (cited from the literature). Almost the same BA was obtained in both 344

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Table 2. Oral BA of Five Drugs in Cynomolgus Monkeys and Humansa cynomolgus monkeys

humansb

compound

dose

BA (%)

dose

BA (%)

acetaminophen atenolol furosemide propranolol naproxen

7.7 mg/kg 1 mg/kg 1 mg/kg 1 mg/kg 1 mg/kg

16 57 32